Combination therapy for treatment of FIV infection

ABSTRACT

The present invention pertains to methods for therapeutic and prophylactic treatment of cats against FIV infection. Methods of the present invention utilize a combination of antiretroviral compounds to treat or prevent FIV infection in a feline animal. In one embodiment, the method comprises administering an effective amount of AZT and another nucleoside analog, such as, for example, 3TC to the animal. In another embodiment, cats are given an effective dose(s) of AZT, 3TC, and a retroviral protease inhibitor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/763,037, filed Jul. 24, 2001, which is the national stage ofinternational application No. PCT/US99/11940, filed May 28, 1999, whichclaims the benefit of U.S. provisional application Ser. No. 60/087,281,filed May 29, 1998, now abandoned.

The subject invention was made with government support under a researchproject supported by NIH Grant AI30904. The government has certainrights in this invention.

BACKGROUND OF THE INVENTION

Feline immunodeficiency virus (FIV) is a lentivirus which causesimmunodeficiency syndrome in domestic cats (Pedersen et al., 1987;Siebelink et al., 1990). FIV closely resembles human immunodeficiencyvirus (HIV) in genomic, biochemical, and morphologic characteristics aswell as clinical and hematological manifestations (Johnson et al., 1994;Pedersen et al., 1987; Yamamoto, Sparger et al., 1988). As a result, FIVinfection of domestic cats is considered to be an excellent small animalmodel for testing prophylactic and therapeutic strategies against AIDSviruses (Gardner, 1991; Johnson et al., 1994). A number ofantiretroviral drugs for HIV, including the prototype nucleosideanalogue azidothymidine (AZT), has been tested using the FIV model (Hartet al., 1995; Hartmann et al., 1992; Hayes et al., 1993; Hayes et al.,1995; Meers et al., 1993; North et al., 1989; Smyth et al., 1994).

The therapeutic use of AZT has been unremarkable in cats and was unableto delay the spread of FIV infection in vivo (Hart et al., 1995;Hartmann et al., 1992). Prophylactic AZT treatment of experimental catscaused either a delay or decrease in both infected blood lymphocytenumbers and plasma virus load (Hayes et al, 1993; Hayes et al., 1995;Meers et al., 1993; Smyth et al., 1994). In addition, a delay in FIVantibody production was observed in some animals (Smyth et al., 1994).However, prophylactic therapy with AZT did not protect cats from FIVinfection (Meers et al., 1993; Hayes et al., 1993; Hayes et al., 1995;Smyth et al., 1994). As reported for HIV therapy, withdrawal of the drugresulted in a resurgence of the virus in these cats. When compared tothe untreated group, significantly higher CD4 and CD8 cell counts wereobserved shortly after the withdrawal of the drug (Hayes et al., 1993;Hayes et al., 1995). However, CD4/CD8 ratios were not significantlydifferent from the untreated cats. In contrast, FIV-infected catstherapeutically treated with AZT had no change in FIV antigen oranti-FIV antibody titers but had transient improvement in CD4/CD8 ratiosand clinical signs (Hart et al., 1995; Hartmann et al., 1992). Thesefindings suggest that monotherapy with AZT has limited benefit as atherapy for FIV infection. Similar observations have been made with AZTmonotherapy of HIV-infected individuals (Harrigan, 1995; Staszewski,1995).

In recent trials, combination therapies with AZT and otherantiretroviral drugs, such as phosphonomethoxyethyl) adenine anddideoxycytidine 5′-triphosphate, had minimal to no effect in preventingor controlling FIV infection in cats (Hartmann et al., 1992; Magnani etal., 1994; Philpott et al., 1992). The in vivo use of viral proteaseinhibitors or new nucleoside analogue combinations, such as, forexample, lamivudine (3TC) and AZT has yet to be reported in FIV-infectedcats. Commercially available HIV protease inhibitors (e.g., Sequinavir(SQV), Indinavir (IDV), Ritonavir, Nelfinavir) do not inhibit FIVreplication in PBMC in vitro. Unlike other nucleoside analogues, 3TCrapidly induces mutations which can phenotypically reverse the mutationscaused by AZT, enabling the antiviral activity of AZT to persist in thehost (Boucher et al., 1993; Larder, 1995; Tisdale et al., 1993). Thisunique feature of 3TC makes it a prime candidate for use in combinationwith AZT. In HIV-positive individuals, the combination AZT/3TC therapyhad synergistic or additive effects at decreasing plasma virus load andincreasing CD4 cell counts and function (Katlama et al., 1994; Lange,1995; Paul et al., 1995; Staszewski, 1995). The addition of an HIVprotease inhibitor to this combination further decreased the viral loadand improved the CD4 cell count (Deeks et al., 1997; Torres et al.,1997).

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns methods for therapeutic and prophylactictreatment of feline animals against infection by FIV. Methods of thepresent invention utilize a combination of antiretroviral compounds. Inone embodiment, an effective amount of a composition comprising AZT andanother nucleoside such as 3TC. In another embodiment, cats are given aneffective dose(s) of a composition comprising AZT, a nucleoside analogsuch as 3TC and a retroviral protease inhibitor. In an exemplifiedembodiment, the protease inhibitor is HBY-793 (Hoescht-Bayer).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows anti-FIV activities of AZT, 3TC, FIV-PI, and HIV-PI (IDVand SQV) in chronically FIV-infected cell lines. The antiviral activityof the drugs at noncytotoxic doses were evaluated in feline T-cell lineschronically infected with either FIV_(Pet) (subtype A strain) (panel A),or FIV_(Bang) (subtype B strain) (panel B). The RT data are presented as% control, whereby % control represents RT mean of triplicate treatedcultures divided by RT mean of triplicate untreated cultures andmultiplied by 100. The RT data on harvest days at 6, 9, and 12 areshown. The results from treated culture sets which are statisticallydifferent from the values of the untreated controls are indicated byeither p<0.05 (P) or p<0.005 (P*) based on Student T test.

FIG. 2 shows anti-FIV activities of AZT, 3TC, FIV-PI, and FIV-PI inprimary PBMC infected with FUV_(Bang). Six separate experiments withvarying concentrations and combinations were performed and the resultsfrom two representative experiments are shown. Nucleoside analogue andPI doses were 0.1 μM in Experiment 1 (panel A) and 0.05 μM and 0.01 μM,respectively, in Experiment 2 (panel B). The RT data are presented as %control and the results from treated culture sets which arestatistically different from the values of the untreated controls areindicated by either p<0.05 (P) or p<0.005 (P*) based on Student T test.The Harvest Day 16 result for AZT/3TC culture set was statisticallydifferent (p<0.03) from the results of AZT culture set and 3TC cultureset from the same time point, as indicated by (Y) above AZT/3TC bar(panel A). The Day 9 and 12 harvest results for AZT/3TC/FIV-PI cultureset were statistically different (p<0.05) from the results of AZT/3TCculture set and FIV-PI culture set from the same time points, asindicated by (Z) above AZT/3TC/FIV-PI bars (panel B).

FIG. 3 shows anti-FIV activities of AZT, 3TC, FIV-PI, and HIV-PI inprimary PBMC infected with FIV_(UK-8)(subtype A strain). Four separateexperiments with varying concentrations and combinations were performedand the results from two representative experiments are shown.Nucleoside analogue and P1 doses were 0.1 μM and 0.01-0.5 μM,respectively, in Experiment 1 (panel A) and 0.05 μM and 0.01-0.5 μMrespectively, in Experiment 2 (panel B). The RT data are presented as %control and the results from treated culture sets which arestatistically different from the values of the untreated controls areindicated by either p<0.05 (P) or p<0.005 (P*). Statistical differencesexisted between the results of AZT/3TC culture set and 3TC culture setat Harvest Days 17 (p<0.02) and 20 (p<0.001) in panel A and Harvest Days9 (p<0.02), 12 (p<0.04), and 15 (p<0.02) in panel B, as indicated by (X)above the AZT/3TC bars. In addition, statistical difference existedbetween the results of AZT/3TC culture set and AZT culture set atHarvest Day 20 (p<0.001) in panel A and Harvest Day 15 (p<0.01) in panelB, as indicated by (Z) above the AZT/3TC bars.

FIG. 4 shows the chemical structure of the protease inhibitor designatedherein as HBY-793.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention concerns methods for therapeutic and prophylactictreatment of cats against infection by FIV. Methods of the presentinvention utilize a combination of antiretroviral compounds. In oneembodiment, cats can be administered an effective amount of acomposition comprising AZT and another nucleoside analog. Preferably,the nucleoside analog is 3TC.

In another embodiment of the methods of the present invention, cats aregiven an effective dose(s) of a composition comprising AZT, anothernucleoside analog and a retroviral protease inhibitor. Preferably, thenucleoside analog is 3TC. In an exemplified embodiment, the proteaseinhibitor is HBY-793. The structure of HYB-793 is shown in FIG. 4. Otherretroviral protease inhibitors that can inhibit FIV proteases arecontemplated within the scope of this invention.

FIV-infected cats treated according to the methods of the presentinvention can also be given bone marrow transplantation after total bodyirradiation in conjunction with the antiretroviral drug combinationtherapy. The bone marrow transplanted can be either allogeneic orautologous.

The antiretroviral compositions of the subject invention can beadministered using standard procedures known in the art. For example,the compositions can be administered as oral or nasal formulations. Thecompositions can also be administered by parenteral injection, i.e.,intravenous, intramuscular, or subcutaneous injection. The amounts anddosage regimens for administration can readily be determined by theordinarily skilled clinician.

Cats that are not infected with FIV can be treated according to themethods of the present invention to provide effective prophylactictreatment against FIV infection. FIV-infected cats can be treatedaccording to the subject methods to provide effective therapy forcontrolling, inhibiting or eliminating FIV infection in that cat.

Results from studies described herein show that the addition of anucleoside analog like 3TC to prophylactic AZT therapy will completelyprotect cats against FIV infection. This observation is supported by thein vitro findings demonstrating that an AZT/3TC combination was moreeffective at inhibiting FIV replication in PBMC cultures thansingle-drug treatments using AZT or 3TC alone. The AZT/3TC combinationis effective when used prophylactically or immediately upon FIVexposure. In addition, the combination of antiretroviral drugsAZT/3TC/FIV-PI can be used as an anti-FIV therapy to treat chronicallyinfected animals.

The present invention also concerns kits comprising in one or morecontainers AZT, another nucleoside analog and an inhibitor of aretroviral protease. Preferably, the nucleoside analog is 3TC. In apreferred embodiment, the retroviral protease inhibitor is HBY-793.

The following abbreviations of FIV strains are used herein: Strain(subtype) Abbreviation Petaluma (A) FIV_(pet) Dixon (A) FIV_(Dix) UK8(A) FIV_(UK-8) Bangston (B) FIV_(Bang) Aomori-1 (B) FIV_(Aom1) Aomori-2(B) FIV_(Aom2) Shizuoka (D) FIV_(shi)

All references cited herein are incorporated by reference.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 In Vitro Efficacy of AZT, 3TC, and PI

In the first set of in vitro studies, feline T-cell lines chronicallyinfected with FV_(Pet) (FL-4 cells) or FIV_(Bang) (FIV_(Bang)/FeT-Jcells) at 2×10⁵ cells/ml were treated for 3 weeks with a single drug orvarious combinations of AZT, 3TC, an FIV protease inhibitor (FIV-PI;Hoescht-Bayer HBY-793), and HIV protease inhibitors (HIV-PI) (FIGS. 1Aand 1B). Saquinavir (SQV) and Indinavir (IDV) were used as the HIV-PIs.Culture supernatants were harvested and the cells were resuspended infresh culture media containing appropriate drug(s) at 34 day intervals.Viral replication was determined by measuring the levels of reversetranscriptase (RT) activity in the culture supernatants (Rey et al.,1984). Drug toxicity in these cultures were monitored by viability andabsolute cell count analyses using trypan blue exclusion method (Mishellet al., 1980). Single and combination drug doses which were determinedto be nontoxic to the test cells were used in these studies.

Both single and combination treatments with AZT and 3TC had minimal tono effect at inhibiting RT activity in FIV_(Bang)/FeT-J cells (20-50%inhibition) and FL-4 cells (0-10% inhibition). In contrast, FIV-PItreatment inhibited FIV replication by 70-80% in both cell lines (FIGS.1A and 1B). However, the addition of an AZT/3TC combination did notenhance this inhibition. Furthermore, neither SQV nor IDV alone hadsignificant anti-FIV effect (FIGS. 1A and 1B). The differences inanti-FlV activities of these nucleoside analogues and FIV-PI may be dueto the differences in the mechanism(s) of their antiviral activities.AZT and 3TC exert their antiretroviral activity by preventing thereverse transcription of viral RNA into viral DNA, whereas FIV-PIprevents the production of a whole virion by inhibiting the FlV proteasefrom cleaving viral gag-pro-pol precursor into their individualcomponents. Therefore, cell lines which have proviral integration willnot be affected by nucleoside analogues. Based on semi-quantitative PCRanalysis, FIV_(Bang)/FeT-J cells and FL-4 cells used in current studyhad proviral integration of 50-80% and >95%, respectively (data notshown). The minor anti-FIV activity of AZT and 3TC observed inFIV_(Bang)/FeT-J cells may be due to the antiviral effect of thenucleoside analogues on the 20-50% of the cells which were still free ofFIV proviral integration. As expected, potent anti-FIV activity wasobserved with FIV-PI in both proviral integrated cell lines.

As a means to simulate in vivo conditions, primary peripheral bloodmononuclear cells (PBMC) from specific pathogen free (SPF) cats werenext used as the indicator cells. Primary PBMC isolated by ficollhypaque method were stimulated with concanavalin A for 3 days andcultured for an additional 2 weeks before their use in drug studies(Staszewski, 1995). Antiretroviral drug(s) were added to the PBMCcultures (1×10⁶ cells/ml) immediately before FIV_(Bang) (subtype B) orFIV_(UK-8) (subtype A) inoculation of 100 50% tissue culture infectiousdose (TCID₅₀). Both single and combination treatments with AZT and 3TCinhibited the FIV replication in PBMC at doses which were not toxic tothe cells (FIGS. 2A and 3A). Synergism in antiviral activities ofAZT/3TC combination was observed against both FIV_(Bang) and FIV_(UK-8)strains (FIGS. 2A, 3A, and 3B). The addition of the FIV-PI to theAZT/3TC combination further enhanced the activities of these drugsagainst FIV_(Bang) (FIG. 2B). Such enhancement was not observed againstFIV_(UK-8) at the doses used (FIGS. 3A and 3B). Thus, the anti-FIVactivities of AZT, 3TC, and FIV-PI are not restricted to specific FIVstrain or subtype, although some strains appear to be more sensitive toone drug over another. Similar to previous studies with chronicallyinfected cells, single-drug treatments with FIV-PI but not HIV-PIs (SQVand IDV) inhibited FIV replication in PBMC cultures (FIGS. 2A, 3A, and3B). Furthermore, addition of SQV or IDV to the AZT/3TC combination didnot enhance the antiviral activity of the AZT/3TC combination. The lackof anti-FIV activity of SQV and IDV may be explained by the fact thatHIV-PIs do not efficiently bind to FIV protease, whereas the FIV-PI usedin this study efficiently binds to HIV protease as well as FIV protease(Dunn et al., 1994; Wlodawer et al., 1995). These results show that dualand triple combinations of AZT, 3TC, and FIV-PI may have therapeuticbenefit against FIV infection in domestic cats.

Example 2 Prophylactic Efficacy of AZT/3TC in Cats

Based on the findings from in vitro studies, the prophylactic use ofAZT/3TC combination was next tested in experimental cats. Four of theeight SPF cats (16-20 weeks of age) received oral administration of AZTand 3TC (75 mg/kg each) twice a day (BID), while remaining cats receivedplacebo. This treatment dose was based on the in vivo research, in whichsix SPF cats (2 cats per treatment group) treated (BID) with either AZTor 3TC at 100 mg/kg or AZT/3TC combination at 50 mg/kg each had nohematological or clinical abnormalities after two weeks of treatment. Inthis study, all cats except for one treated cat (#RU1) were inoculatedwith 100 50% cat infectious dose (C1D₅₀) of FIV_(UK-8) at 3 days afterthe first drug or placebo treatment. FIV_(UK-8) was used in this studybecause this strain gave more consistent CD4/CD8 ratio inversion in alarger number of infected cats than did infection with FIV_(Bang) orFIV_(Pet). All cats received either the drug or placebo treatmentsthroughout the first 11 weeks after FIV inoculation, unless statedotherwise. The cats were monitored daily for clinical signs and twice amonth for hematological changes, FIV load in PBMC and plasma, anti-FIVantibody titers, and CD4/CD8 ratio and absolute counts (Diehi et al.,1995; Green et al., 1993; Okada et al., 1994; Tellier et al., 1997;Yamamoto et al., 1991).

At 4 weeks of treatment, severe anemia was observed in all challengedand unchallenged cats treated with AZT/3TC; therefore, the doses of eachdrug were lowered to 34 mg/kg each at 4 weeks of treatment andsubsequently to 5-10 mg/kg each at 5 weeks of treatment. AZT/3TCtreatment was terminated in one cat (#3 GB) at 6 weeks of treatment, andthe treatment was resumed 6 days later at 5 mg/kg each. Based on virusisolation and PCR analyses, one cat (#101) from the placebo group waspositive for FIV by 3 weeks post infection (pi) and had anti-FIVantibodies by 5 weeks pi (Table 1). However, plasma viral RNA levels ofthis cat were not detected throughout the study; even though the virusload in the PBMC was similar to the levels detected in the remainingplacebo cats. These placebo cats (#NK4, #NK6, #IH5) were positive forFIV titers in the plasma and PBMC and for anti-FIV antibodies by 7 weekspi. Furthermore, all placebo cats, except for cat #101, had transient orpersistent CD4/CD8 inversion starting 11 weeks pi. In contrast, allAZT/3TC-treated cats were negative for FIV and had no CD4/CD8 inversionthroughout the study. Both drug and placebo treatments were terminatedat 11 weeks pi and all cats were monitored for additional 6-13 weeks. Inthe previous reports, an increase in FIV load of the PBMC was observedafter the withdrawal of AZT treatment in FIV-infected cats (Hayes, etal., 1993; Hayes et al., 1995; Meers et al., 1993). Thus, if low levelsof FIV infection undetectable by current assays existed inAZT/3TC-treated cats, then such infection should rebound when the drugsare removed. In this study, all AZT/3TC-treated cats remained negativefor FIV in PBMC and anti-FIV antibodies throughout the 6-13 weeks afterthe withdrawal of the drug treatment. Virus isolation and PCR of bonemarrow and lymph node cells performed at the termination of the studyfurther confirmed the FIV-free status of these cats. Thus, completeprotection of cats against experimental FIV infection was achieved withprophylactic AZT/3TC therapy.

Example 3 Therapeutic Efficacy of AZT/3TC in Chronically FIV-InfectedCats

Based on the in vivo toxicity observed in the prophylactic study, threecats (#101, #NK6, #144) chronically infected with FIV_(UK-8) for 16weeks were treated at 20 mg/kg of each drug (BID), while an additionalthree infected cats (#1H5, #NK4, #158) received placebo. These cats weretreated with either drug combination or placebo for 8 weeks andmonitored an additional 4 weeks for changes in FIV load and CD4/CD8values. All parameters monitored were identical to those of theprophylactic study. All treated cats developed either mild or severeanemia by 3.5 weeks of treatment. As a result, both drug doses werelowered to 10 mg/kg. Nevertheless, the anemia in one cat (#144) becameso severe by 6 weeks of treatment that the drug treatment was terminatedfor 1 week and resumed thereafter at a low dose of 5 mg/kg of each drug(BID). Unlike the prophylactic study, no significant differences ineither FIV load or CD4/CD8 ratios and absolute counts were observedbetween the treated and placebo cats (Table 2). This study incombination with the previous studies suggest that doses even as low as20 mg/kg of each drugs when used over moderate period of time (3.5 weeksor longer) will cause anemia in cats. However, short-term treatment (2weeks) with high dose combination (75 mg/kg each) is well tolerated bycats.

Example 4

Allogeneic bone marrow transplantation (BMT) in combination with totalbody irradiation (TBI) and anti-FIV drug therapy was evaluated as animmune reconstitution therapy for FIV-infected cats. The rationale forthis therapy is as follows: (1) TBI will decrease FIV load by destroyingrecipient's hematopoietic cells, including FIV-infected immunocytes. (2)Anti-FIV drug therapy can block the infection of engrafted donor cellsin the BMT recipients. (3) BMT with donor BM cells from uninfected catswill reconstitute normal hematopoietic system. The TBI/BMT combinationalone was unable to decrease the virus load due to rapid infection ofengrafted donor cells. A majority of FIV-infected recipients ofallogeneic BMT succumbed to graft-versus-host disease, acceleratedFIV-related diseases, or their combination. As a result, studies wereperformed to identify antiretroviral drugs that can be combined withTBI/BMT. Prophylactic therapy with AZT/3TC combination protected 100% ofthe cats from FIVUK 8 challenge infection. Moreover, the onlyFIV-infected cat to survive allogeneic BMT also received concurrentAZT/3TC therapy. This cat had complete hematopoietic engraftmentincluding normal CD8 counts. However, its CD4 counts were only slightlyhigher than the levels observed before BMT. Furthermore, only slightdecrease in plasma virus load was observed during high-dose AZT/3TCtherapy. Nonetheless, its anti-FIV antibody titers were 100-fold lowerthan those before BMT. This cat was still healthy at one year post-BMTand is still responsive to AZT/3TC therapy.

Example 5

Recent findings with anti-HIV triple-drug combination have revealed thattriple-drug cocktails are unable to immune reconstitute the patient withnormal numbers and repertoire of T cell populations or to completelydecrease/remove the virus load in the lymphoid tissues within feasibleduration of time. As such, autologous bone marrow transplantation (BMT)was tested in combination with antiretroviral drugs as an immunereconstitution therapy for FIV-infected cats. Based on preliminaryresults, no significant decrease in FIV load or improvement in CD4/CD8ratios or counts were detected in infected cats that received autologousBMT one (1) day after total body irradiation (TBI). These cats survivedthe autologous BMT and are currently alive over two years after BMT.This is in contrast to the results from allogeneic BMT of FIV-infectedcats, whereby all cats except the one on AZT/3TC therapy succumbed toGVHD, accelerated FIV-disease, or their combination. The extension ofthe time of BMT after TBI will decrease the infected cell reservoir loadand, consequently, fewer infected cells will be available to infectengrafted cells. Addition of antiretroviral drug therapy will preventany remaining infected cell reservoir from contaminating the engraftedcells.

Example 6 Pharmaceutical Compositions

Antiviral compounds of the invention can be formulated according toknown methods for preparing pharmaceutically useful compositions.Remington's Pharmaceutical Science by E. W. Martin describesformulations which can be used in connection with the subject invention.In general, the compositions of the subject invention will be formulatedsuch that an effective amount of the antiviral compounds are is combinedwith a suitable carrier in order to facilitate effective administrationof the composition. It should, of course, be understood that thecompositions and methods of this invention may be used in combinationwith other therapies.

The compositions used in these therapies may also be in a variety offorms. These include, for example, solid, semi-solid, and liquid dosageforms, such as tablets, pills, powders, liquid solutions or suspensions,liposomes, suppositories, injectable, and infusible solutions. Thepreferred form depends on the intended mode of administration andtherapeutic application. The compositions also preferably includeconventional pharmaceutically acceptable carriers and adjuvants whichare known to those of skill in the art. Preferably, the compositions ofthe invention are in the form of a unit dose.

Once improvement in condition has occurred, a maintenance dose isadministered if necessary. Subsequently, the dosage or the frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. TABLE 1 AZT/3TCprophylaxis of cats starting 3 days before FIV inoculation AZT/3TCtreatment^(a) (kg/mg) FIV levels^(b) −0.4→4→5→6-7 FIV VI/PCR/vRNA FIVantibodies^(b) CD4/CD8 ratio^(bc) Cat # wk inocul. Pre 4 wk 9 wk 11 wk14 wk Pre 4 wk 9 wk 11 wk 14 wk Pre 7 wk 11 wk 14 wk DH5 75→34→10 +−/−/− −/−/− −/−/− −/−/− −/−/− − − − − − 3.30 2.86 2.62 2.38 3GB75→34→10→0-5 + −/−/− −/−/− −/−/− −/−/− −/−/− − − − − − 1.56 1.37 1.371.47 RU2 75→34→10 + −/−/− −/−/− −/−/− −/−/− −/− − − − − − 1.77 1.21 1.181.18 RU1 75→34→10 − −/−/− −/−/− −/−/− −/−/− −/−/− − − − − − 2.37 1.621.62 1.47 NK4 — + −/−/− +/+/+ +/+/+ +/+/+ +/+/+ − − + + + 1.82 1.55 0.960.91 NK6 — + −/−/− −/+/+ +/+/+ +/+/+ +/+/+ − − + + + 1.61 0.92 0.46 0.45IO1 — + −/−/− +/+/− +/+/− +/+/− +/+/− − − + + + 3.40 1.73 1.24 1.23 IH5— + −/−/− −/+/+ +/+/+ +/+/+ +/+/+ − − + + + 4.40 1.34 0.60 0.61^(a)The AZT/3TC treatment was started 3 days before FIV inoculation(−0.4 post-infection) at a dose of 75 mg/kg each and decreased to 34mg/kg at 4 wk post-infection (pi) and then to 10 mg/kg at 5 wk pi. Inone cat (#3GB), the AZT/3TC treatment was withdrawn at 6 wk pi andresumed at a low dose of 5 mg/kg at# 7 wk pi. The changes in doses of each drug, including the amount(mg/kg) and time (wk pi), are shown.^(b)Samples before drug or placebo treatment (Pre) and those at variousweeks post-infection (wk) were tested for FIV levels, FIV antibodies,and CD4/CD8 rations. FIV levels were determined by virus isolation (VI),PCR for FIV provirus in PBMC, and RT-PCR for plasma viral RNA (vRNA).FIV antibodies were determined# by immunoblot analysis. In general, RT-PCR for plasma viral RNA wasless sensitive than PCR of FIV provirus in PBMC after amplification ofinfected cells by coculturing.^(c)Inverted CD4/CD8 ratios are bolded.

TABLE 2 AZT/3TC therapy of FIV-infected cats FIV load^(b) AZT/3TCtreatment^(a) (No. of infected (kg/mg) cells in PBMC) FIV antibodies^(b)CD4/CD8 ratio^(b) Cat No. 0 wk→3.5 wk→6-7 wk 0 wk 3.5 wk 8 wk 12 wk 0 wk3.5 wk 8 wk 12 wk 0 wk 3.5 wk 8 wk 12 wk 101 20→10 ++ + + ++ + + ++ ++1.45 1.58 1.71 1.71 NK6 20→10 ++ ++ + + ++ ++ ++ ++ 0.45 1.00 0.64 0.70144 20→10→0-5 +++ ++++ +++ ++ ++ ++ ++ ++ 0.67 0.74 0.45 0.52 IH5 — ++++ + ND ++ ++ ++ ++ 0.61 0.63 0.73 0.87 NK4 — ++ + + + + ++ ++ ++ 0.911.25 1.54 1.54 158 — +++ +++ +++ ++ + ++ ++ ++ 1.08 1.01 1.33 1.06^(a)The doses of each drug were decreased from 20 mg/kg to 10 mg/kg at 3weeks treatment. Treatment was withdrawn in one cat (#144) at 6 weeks oftreatment and resumed one week later at 5 mg/kg. The changes in doses ofeach drug, including amount (mg/kg) and time (wk after initialtreatment), are shown.^(b)Samples before drug or placebo treatments (0 wk) and those atvarious weeks after initial treatment (wk) were tested for FIV levels,FIV antibodies, and CD4/CD8 ratios. FIV loads were determined by thenumber of PBMC (50 to 5 × 10⁶ PBMC cocultured with 5 × 10⁶ feeder PBMC)needed for positive virus# isolation. Virus isolation results are presented as 50 (++++), 5 × 10²(+++), 5 × 10³ (++), and 5 × 10⁴ (+) PBMC from treated and untreatedcats needed to isolate FIV from a culture containing 5 × 10⁶ uninfectedfeeder PBMC. FIV antibody titer is defined as the minimal dilution (inLog₁₀) at which antibodies to FIV major # core protein (p26) could bedetected. Serial log dilutions of serum (10⁻⁴ to 10⁻⁷ dilution) wereincubated with immunoblot strip for 2 hrs and processed using theimmunoblot method. End point titrations of FIV antibodies are presentedas 10⁻⁵ (+) and 10⁻⁶ (++).

REFERENCES

-   Boucher, C. A. B., N. Cammack, P. Schipper, R. Schuurman P.    Rouse, M. A. Wainberg, and J. M. Cameron (1993) “High-level    resistance to (−) enantiomeric 2-deoxy-3 thiacytidine in vitro is    due to one amino acid substitution in the catalytic site in human    immunodeficiency virus type I reverse transcriptase” Antimicrob.    Agents Chemother. 37: 2231-2234.-   Deeks, S. G., M. Smith, M. Holodniy, J. O. Kahn (1997) “HIV-1    protease inhibitors” J. Am. Med. Assoc. 277: 145-153.-   Diehi, L. J., C. K. Mathiason-Dubard, L. L. O'Neil, and E. A.    Hoover (1995) “Longitudinal assessment of feline immunodeficiency    virus kinetics in plasma by use of a quantitative competitive    reverse transcriptase PCR” J. Virol. 69: 2328-2332.-   Dunn, B. M., A. Gustchina, A. Wlodawer, and J. Kay (1994) “Subsite    preferences of retroviral proteinases” Meth. Enzymol. 241: 254-278.-   Gardner, M. B. (1991) “Mini-review. Simian and feline    immunodeficiency viruses: animal lentivirus models for evaluation of    AIDS vaccines and antiviral agents” Antiviral Res. 15: 267-286.-   Green, W. K, J. Meers, G. del Fierro, P. R. Carnegie, and W. F.    Robinson (1993) “Extensive sequence variation of feline    immunodeficiency virus env genes in isolates from naturally infected    cats” Arch. Virol. 133: 51-62.-   Harrigan, R. (1995) “Measuring viral load in the clinical    setting” J. Acquir. Immune Defic. Syndr. Hum Retrovirol. 10(Suppl.    1): S34-S40.-   Hart, S., and I. Nolte (1995) “Long-term treatment of diseased,    FIV-seropositive field cats with azidothymidine (AZT)” J. Vet.    Med. A. 42: 397-409.-   Hartmann, K, A. Donath, B. Beer, H. F. Egberink, M. C. Horzinek, H.    Lutz, G. Hoffmann-Fezer, I. Thum, and S. Thefeld (1992) “Use of two    virustatica (AZT, PMEA) in the treatment of FIV and of FeLV    seropositive cats with clinical symptoms” Vet. Immunol.    Immunopathol. 35: 167-175.-   Hayes, K. A., L. J. Lafrado, J. G. Erickson, J. M. Marr, and L. E.    Mathes (1993) “Prophylactic ZDV therapy prevents early viremia and    lymphocyte decline but not primary infection in feline    immunodeficiency virus-inoculated cats” J. Acquir. Immune Defic.    Syndr. 6: 127-134.-   Hayes, K. A., J. G. Wilkinson, R. Frick, S. Francke, and L. E.    Mathes (1995) “Early suppression of viremia by ZDV dose not alter    the spread of feline immunodeficiency virus infection in cats” J.    Acquir. Immune Defic. Syndr. Hum. Retrovirol. 9: 114-122.-   Johnson, C. M., B. A. Torres, H. Koyama, and J. K. Yamamoto (1994)    “FIV as a model for AIDS vaccination” AIDS Res. Hum. Retroviruses    10: 225-228.-   Katlama, C., and the European Lamivudine HIV Working Group (1994)    “Combination 3TC (lamivudine)/ZVD (zidovudine) versus ZVD    monotherapy in ZVD naive HTV-1 positive patients with CD4 of 100-40    Ω cells/mm3, abstr” AIDS 8(Suppl. 4): 56.-   Lange, J. M. A. (1995) “Triple combinations: present and future” J.    Acquir. Immune Defic. Syndr. Hum. Retrovirol. 10(Suppl. 1): S77-S82.-   Larder, B. A. (1995) “Viral resistance and the selection of    antiretroviral combinations” J. Acquir. Immune Defic. Syndr. Hum.    Retrovirol. 10(Suppl. 1): S28-S33.-   Magnani, M., L. Rossi, A. Fraternale, L. Silvotti, F.    Quintavalla, G. Piedimonte, D. Matteucci, F. Baldinotti, and M.    Bendinelli (1994) “Feline immunodeficiency virus infection of    macrophages: In vitro and in vivo inhibition by    dideoxycytidine-5′-triphosphate-loaded erythrocytes” AIDS Res. Hum.    Retroviruses 10: 1179-1186.-   Meers, J., G. M. del Fierro, I L B. Cope, H. S. Park, W. K Greene,    and W. F. Robinson (1993) “Feline immunodeficiency virus infection:    plasma, but not peripheral blood mononuclear cell virus titer is    influenced by zidovudine and cyclosporine” Arch. Virol. 132: 67-81.-   Mishell, B. B., S. M. Shilgi, C. Henry, E. L. Chan, I. North, R.    Gallily, M. Slomich, K Miller, I. Marbrook, D. Parks, and A. H.    Good (1980) “Preparation of mouse cell suspensions” p. 3-27.    In B. B. Mishell and S. M. Shiigi (ed.), Selected methods in    cellular immunology. W.H. Freeman and Co. San Francisco, Calif.-   Newell, M. L., and D. M. Gibb (1995) “A risk-benefit assessment of    zidovudine in the prevention of perinatal HIV trarisnussion”    Drug-Saf: 12: 274-281.-   North, T. W., G. L. T. North, and N. C. Pedersen (1989) “Feline    immunodeficiency virus, a model for reverse transcriptase-targeted    chemotherapy for acquired immunedeficiency syndrome” Antimicrob.    Agents Chemother. 33: 915-919.-   Okada, S., R. Pu, E. Young, W. Stoffs, and J. K. Yamamoto (1994)    “Superinfection of cats with FIV subtypes A and B” AIDS Res. Hum.    Retroviruses 10: 1739-1746.-   Paul, D. B., M. C. Kuhns, A. L. McNamara, J. C. Jr. Pottage,    and G. T. Spear (1995) “Short-term stability of HIV provirus levels    in the peripheral blood of HIV-infected individuals” J. Med. Virol.    47: 292-297.-   Pedersen, N. C., E. W. Ho, M. L. Brown, and J. K. Yamamoto (1987)    “Isolation of a T-lymphotropic virus from domestic cats with an    immunodeficiency-like syndrome” Science 235: 790-793.-   Philpott, M. S., J. P. Ebner, and E. A. Hoover (1992) “Evaluation of    9-(2-phosphonylmethoxyethyl) adenine therapy for feline    immunodeficiency virus using a quantitative polymerase chain    reaction” Vet. Immunol Immunopathol. 35: 155-166.-   Rey, M. A, B. Spire, D. Dormont, F. Barre-Sinoussi, L. Montagnier,    and J. C. Chermann (1984) “Characterization of the RNA dependant DNA    polymerase of new human T-lymphotropic retrovirus (lymphadenopathy    associated virus)” Biochem. Biophys. Res. Commun. 21: 1247-1253.-   Siebelink, K. H. J., I-H, Chu, G. F. Rimmelzwaan, K. Weijer, R. V.    Herwijnen, P. Knell, H. F. Egberink, M. L. Bosch, and A. D. M. E.    Osterhaus (1990) “Feline immunodeficiency virus (FIV) infection in    the cats as a model for HIV infection in man: FIV-induced impairment    of immune function” AIDS Res. Hum. Retroviruses 6: 1373-1378.-   Smyth, N. R., M. Bennett, R. M. Gaskell, C. M. McCracken, C. A.    Hart, and J. L. Howe (1994) “Effect of 3′azido-2′3′-deoxythymidine    (AZT) on experimental feline immunodeficiency virus infection in    domestic cats” Res. Vet. Sci. 57: 220-224.-   Staszewski, S. (1995) “Zidovudine and lamivudine: results of phase    Ill studies” J. Acquir. Immune Defic. Syndr. Hum Retrovirol.    10(Suppl. 1): 557.-   Tellier, M. C., J. M. Soos, R. Pu, and J. K. Yamamoto (1997)    “Development of FIV-specific cytolytic T-lymphocyte responses in    cats upon immunization with FIV vaccines” Vet. Microbiol. 57: 1-11.-   Tisdale, M., S. D. Kemp, N. R. Parry, and B. A. Larder (1993) “Rapid    in vitro selection of human immunodeficiency virus type I resistant    to 3-thiacytidine inhibitors due to a mutation in the YMDD region of    reverse transcriptase” Proc Natl. Acad. Sci. USA 90: 5653-5656.-   Torres, R. A. and M. R. Barr (1997) “Combination antiretroviral    therapy for HIV infection” Infect. Med. 14: 142-160.-   Wlodawer, A., A. Gustchina, L. Reshetnikova, J. Lubkowski, A.    Zdanov, K. Y. Hui, E. L. Angleton, W. G. Farmerie, M. M.    Goodenow, D. Bhatt, L. Zhang, and B. M. Dunn. (1995) “Structure of    an inhibitor complex of the proteinase from feline immunodeficiency    virus” Nature Struct. Biol. 2: 480-488.-   Yamamoto, J. K., T. Okuda, C. D. Ackley, H. Loule, H. Zochlinski, E.    Pembroke, and M. B. Gardner (1991) “Experimental vaccine protection    against feline immunodeficiency virus” AIDS Res. Hum. Retroviruses    7: 911-922.-   Yamamoto, J. K., N. C. Pedersen, E. W. Ho, T. Okuda, and G. H.    Theilen (1988) “Feline immunodeficiency syndrome—A comparison    between feline T-lymphotropic lentivirus and feline leukemia virus”    Leukemia 2(Suppl. 12): 204S-215S.-   Yamamoto J. K., E. Sparger, E. W. Ho, P. R. Andersen, T. P.    O'Connor, C. P. Mandell, L. Lowenstine, R. Munn, and N. C.    Pedersen (1988) “Pathogenesis of experimentally induced feline    immunodeficiency virus infection in cats” Am. J. Vet. Res. 49:    1246-1258.

1. A method for treating or preventing infection of felineimmunodeficiency virus (FIV) in a feline animal, said method comprisingadministering to said feline animal an effective amount ofazidothymidine (AZT) and another nucleoside analog.
 2. The methodaccording to claim 1, wherein said another nucleoside analog is 3TC. 3.The method according to claim 1, wherein said feline animal receivesbone marrow transplantation after total body irradiation.
 4. The methodaccording to claim 3, wherein the transplanted cells are selected fromthe group consisting of allogeneic cells and autologous cells.
 5. Amethod for treating or preventing infection of feline immunodeficiencyvirus (FIV) in a feline animal, said method comprising administering tosaid feline animal an effective amount of azidothymidine (AZT), anothernucleoside analog and an inhibitor of a retroviral protease.
 6. Themethod according to claim 5, wherein said another nucleoside analog is3TC.
 7. The method according to claim 5, wherein said inhibitor of aretroviral protease is selected from the group consisting of HIVprotease inhibitors and FIV protease inhibitors.
 8. The method accodingto claim 5, wherein said inhibitor of a retroviral protease isdesignated as HBY-793 and has the structure shown in FIG.
 4. 9. Themethod according to claim 5, wherein said another nucleoside analog is3TC and said inhibitor of a retroviral protease is designated as HBY-793and has the structure shown in FIG.
 4. 10. The method according to claim5, wherein said feline animal receives bone marrow transplantation aftertotal body irradiation.
 11. The method according to claim 10, whereinthe transplanted cells are selected from the group consisting ofallogeneic cells and autologous cells.
 12. A kit comprising in one ormore containers AZT, another nucleoside analog and an inhibitor of aretroviral protease.
 13. The kit according to claim 12, wherein saidanother nucleoside analog is 3TC.
 14. The kit according to claim 12,wherein said inhibitor of a retroviral protease is designated as HBY-793and has the structure shown in FIG.
 4. 15. The kit according to claim12, wherein said another nucleoside analog is 3TC and said inhibitor ofa retroviral protease is designated as HBY-793 and has the structureshown in FIG. 4.